NEUROTROPHIC AND IMMUNOLOGIC EFFECTS OF RIBOFLAVIN ON MOTOR DISABILITY BUT NOT SPATIAL LEARNING AND MEMORY CONSOLIDATION IN EXPERIMENTAL AUTOIMMUNE ENCEPHALOMYELITIS MODEL OF MULTIPLE SCLEROSIS

Background and Aim: Multiple sclerosis (MS) is an inflammatory demyelinating disease of the central nervous system (CNS) leading a considerable reduction of disabilities in activities of daily and cognitive impairment (1). Experimental autoimmune encephalomyelitis (EAE) is a commonly employed animal model of MS (2). Several studies report the critical pathogenic role of inflammatory cytokines (2) and neurotrophic factors (3) in the pathogenesis of MS/EAE. Riboflavin selectively interferes in the peripheral nerve myelin synthesis (4). In this experimental study, we assessed the effects of riboflavin on motor disability as well as spatial learning and memory in EAE model (5). Methods: Ten-week-old C57BL/6 female adult mice were assigned into 7 groups: sham operated 1 (SO1), healthy mice received PBS (phosphate buffer saline); sham operated 2 (SO2), healthy mice received PBS and riboflavin; sham treatment1 (ST1), EAE mice received water; sham treatment 2 (ST2), EAE mice received sodium acetate buffer; treatment 1 (T1), EAE mice received interferon beta-1a (INFβ-1a); treatment 2 (T2), EAE mice received riboflavin; treatment 3 (T3), EAE mice received INFβ-1a and riboflavin. After EAE induction, scoring was performed based on clinical signs. By detecting score 0.5, riboflavin at 10 mg/kg of body weight and/or INFβ-1a at 150 IU/g of body weight administration (6) were started for two weeks. Spatial learning and memory were assessed through the Morris Water Maze (MWM) test. The brain and spinal cord levels of brain-derived neurotrophic factor (BDNF), interleukin-6 (IL-6), and interleukin-17A (IL-17A) were measured using real-time PCR and ELISA methods. All procedures were approved by the Ethics Committee for Animal Experimentation at the Ahvaz Jundishapur University of Medical Sciences (AJUMS, NRC-9208). The results in the brain revealed that BDNF mRNA expression increased in the EAE mice treated with the combination of riboflavin and INF-β1a compared to the treated groups with riboflavin (95% CI: 4.1-14.7) or INF-β1a (95% CI: 3-14) (p<0.01). An increase in BDNF levels in the brain of EAE mice treated with the combination of riboflavin and INF-β1a (2463.4 ± 832.8 pg/mg tissue) was evident compared to treated EAE mice with INF-β1a (945 ± 148.1 pg/mg tissue; 95% CI: 63.5-2973.1) and riboflavin (837.6 ± 313.7 pg/mg tissue; 95% CI: 231.7-3020) (p< 0.05). Moreover, IL-6 and IL-17A expression were increased in the brains of the T3 and T1, respectively, compared to other groups (p<0.01). Daily clinical score was reduced significantly by riboflavin in both effector and chronic phases of the disease compared with that of controls (p<0.05). The data from MWM test revealed that the mice with EAE performed similarly compared to the healthy controlled mice in terms of latency and swimming distance to find the hidden platform. However, T2 and T3 mice swam faster than the ST2 (p< 0.05), T1 (p< 0.05), and ST1 (p< 0.05) mice (5). We had already shown that riboflavin supplementation (10 mg/day) for six months reduced the expanded disability status scale (EDSS) score significantly in patients with MS. However, this reduction also occurred in the MS patients taking placebo. The rate of decrease was non-significantly higher in the riboflavin than in the placebo group (7). Studies have shown that riboflavin is involved in the metabolism of essential fatty acids in the brain lipids (8). In addition, riboflavin has a role as a cofactor for succinate dehydrogenase in the complex II of mitochondrial respiratory chain generating intracellular adenosine triphosphate (ATP) (9). Since, CNS response to circulating neurotrophins depends on the intracellular ATP availability, enhancement of mitochondrion survival and bio-energy with riboflavin increases its responsiveness to BDNF neurotrophine followed by increasing production of myelin in MS (10) suggesting that BDNF mediates the beneficial effect of riboflavin on neurological motor disability (11). In this study, we have shown an increased expression of IL-6 coincided with increased expression of BDNF in the whole brain and spinal cord of EAE mice receiving both riboflavin and INFβ-1a. IL-6 probably has proliferative effect on astrocytes in vitro (12, 13). Also, IL-6 probably promotes survival through inducing BDNF (14). As a result, IL-6 behaves in a neurotrophin-like fashion (15).Conclusion: Our findings showed that riboflavin is capable for suppressing the neurological disability but not spatial learning and memory consolidation mediated by BDNF and IL-6.

Keywords:

Riboflavin , INF-β1a , Experimental autoimmune encephalomyelitis , Brain-derived neurotrophic factor , Memory